Since the phenotype of sickle cell anemia is multigenic, this project strives to define the pleotroplc genes in sickle cell anemia based on up-regulated genes and down-regulated genes detected by microarray technology combining the use of animal models and human subjects. We have reproducible preliminary data on up-regulated and down-regulated genes during adhesion/obstruction by sickle cells. Aim 1, will define tissue and cellular specificity (mesenterium vs cecum) of the expression of genes in an ex vivo microcirculator preparation: by time-dependence of the patterns ofgene expression, by histochemistry, by state-of-the-art TEM microscopy and by Western blots, to dissect the cell type and tissue involved in up regulation or down regulation. In addition, we will pre-treat the preparation with pharmacological expression-enhancers or ameliorators of the genes of interest and follow by expression profiling. We will expand the exploration of the effect of leukocytes combined with sickle cells in microcirculatory vasocclusion and breed transgenic mice that over-express or under-express the genes of interest (hemizygote or homozygotes) into our sickle mice to enable further definition of the pathophysiology involved. Aim 2, involves the dissection of potential epistatic genes, among the pleiotropic genes involved in sickle cell-mediated vasocclusion, by studying the human polymorpbhsms already established (or searching for new polymorphisms) in the up and down-regulated set of genes. Finally, we will study the effect of these up or down-regulated genes by breeding into sickle mouse models, other transgenic lines in which these genes are altered. Aim 3 addresses a novel issue: while considerable data have been generated to tie the phenotypic effects of the sickle gene in linkage disequilibrium with four different beta-gene cluster haplotypes, little is known about the mechanisms involved. We intend to use a combination of genomic sequence analysis with precise functional testing of DNA sequence elements to explore the impact of "junk" DNA on this phenomena. This effort is likely to greatly improve our understanding of the pathophysiology of sickle cell anemia and define some of the epistatic genes that impinge on its diverse inter-individual severity.